Podcasts about cytoplasmic

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546610v1?rss=1 Authors: Goncalves, D. L., Peffer, S., Morano, K. A. Abstract: Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1{triangleup} cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a mechanism of long-term sequestration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538370v1?rss=1 Authors: Yonemura, Y., Sakai, Y., Nakata, R., Hagita-Tatsumoto, A., Miyasaka, T., Misono, H. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.07.536038v1?rss=1 Authors: Dougherty, L. L., Avasthi, P. Abstract: At the core of cilia are microtubules which are important for establishing length and assisting ciliary assembly and disassembly; however, another role for microtubule regulation on ciliogenesis lies outside of the cilium. The microtubule cytoskeleton is a highly dynamic structure which reorganizes rapidly to assist in cellular processes. Cytoplasmic microtubule dynamics have previously been thought to be necessary to free up tubulin and proteins in the ciliary precursor pool for ciliogenesis. However, we previously found that low concentrations of taxol can stabilize cytoplasmic microtubules during deciliation while allowing normal cilium regrowth. Here we look at the relationship between ciliogenesis and cytoplasmic microtubule dynamics in Chlamydomonas reinhardtii using chemical and mechanical perturbations. We find that not only can stabilized cytoplasmic microtubules allow for normal ciliary assembly, but high calcium concentrations and low pH-induced deciliation cause microtubules to depolymerize separately from ciliary shedding. In addition, we find that through mechanical shearing, cilia regenerate more quickly despite intact cytoplasmic microtubules. Our data suggests that cytoplasmic microtubules are not a sink for a limiting pool of cytoplasmic tubulin, reorganization that occurs following deciliation is a consequence rather than a requirement for ciliogenesis, and intact microtubules in the cytoplasm and the proximal cilium support more efficient ciliary assembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In this episode, we review the high-yield topic of Nuclear / Cytoplasmic-Acting Hormones ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠from the Endocrine section. Follow ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠Medbullets⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠ on social media: Facebook: www.facebook.com/medbullets Instagram: www.instagram.com/medbulletsofficial Twitter: www.twitter.com/medbullets --- Send in a voice message: https://podcasters.spotify.com/pod/show/medbulletsstep1/message

Dr. Craig Turczynski, the former director of in-vitro fertilization, andrology, and hormone assay laboratories at Louisiana State University Medical Center-Shreveport explains how IVF is done, and why it raises grave moral concerns. CORRECTIONS: 1. The number of IVF procedures annually is about 250,000 not 250. 2. Cytoplasmic transfer is taking cytoplasm from a young woman's egg and injecting it into the infertile woman's egg. It is not sure injected into the woman.  Cy Kellett: Hello and welcome to Focus, the Catholic Answers’ podcast for living, understanding, and defending your Catho…

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.06.519266v1?rss=1 Authors: de Oliveira, M. I. R., Hanson, F. M., Hodgson, R. E., Cross, A. K., Campbell, S. G., Allen, K. E. Abstract: The multisubunit eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) for eIF2, is an essential regulator of translation initiation. Activation of the cellular integrated stress response (ISR) by factors such as endoplasmic reticulum stress leads to phosphorylation of eIF2 and inhibition of eIF2B GEF activity. Cytoplasmic bodies containing eIF2B subunits, termed eIF2B bodies, have been shown to alter in subunit composition and fluorescence recovery after photobleaching activity in response to the ISR. Analysis of the subunit composition of endogenous eIF2B bodies is dependent on accurate detection of each protein in a cellular context via immunocytochemistry (ICC). We describe bioinformatic techniques to optimize the ICC detection of eIF2B foci in U373 cells. The screening of commercially available primary antibodies against predicted epitopes enhanced measurements of the number, size and fluorescence intensity of eIF2B bodies. A consistent and reproducible ICC analysis of endogenous eIF2B bodies will aid characterisation of eIF2B bodies during the ISR or under disease conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.11.516101v1?rss=1 Authors: Querl, L., Lu, Y.-Y., Beissel, C., Krebber, H. Abstract: Accurate translation requires correct mRNAs with intact open reading frames. Cells eliminate defective transcripts to prevent mistranslation by three cytoplasmic mRNA quality control events termed nonsense-mediated decay (NMD), no-go decay (NGD) and non-stop decay (NSD). Translation termination on correct transcripts requires Dbp5 (human DDX19), which delivers eRF1 to the ribosomes and prevents an early contact of eRF1 with eRF3, precluding the immediate dissociation of both release factors and subsequent termination readthrough. Here, we report evidence for an influence of Dbp5 on NMD, as it delivers eRF1 also to PTC-containing transcripts. In contrast to regular translation termination and NMD, functional NGD and NSD require the eRF1-eRF3-like proteins Dom34-Hbs1. We suggest that Dbp5 delivers Dom34 to NGD and NSD substrates as well. However, in contrast to regular termination, it does not prevent an Hbs1 contact, but allows formation of a ternary Dom34-Hbs1-Dbp5 complex. The Dbp5-mediated delivery of Dom34-Hbs1 in NGD and NSD might rather shield and position the complex to prevent a premature contact of Dom34 and Rli1 to prevent inefficient splitting of the ribosomal subunits. Together, we have gathered evidence suggesting an important role of Dbp5 in cytoplasmic mRNA quality control. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513104v1?rss=1 Authors: Zhou, C. Y., Dekker, B., Liu, Z., Cabrera, H., Ryan, J., Dekker, J., Heald, R. Abstract: During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to coordinately scale with mitotic spindles, but underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be re-set by cytoplasmic factors from earlier developmental stages. In vitro, increasing nucleo-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin scales mitotic chromosomes to cell surface area/volume (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and analysis of Hi-C data suggest that mitotic chromosomes scale through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter axis. Together, our findings demonstrate that mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.16.512122v1?rss=1 Authors: Desai, M., Singh, H., Deo, A., Naik, J., Bose, T., Majumdar, A. Abstract: Orb2 the Drosophila homolog of Cytoplasmic polyadenylation element binding protein (CPEB) forms prion-like oligomers. These oligomers consist of Orb2A and Orb2B isoforms and their formation are dependent on the oligomerization of the Orb2A isoform. Drosophila with a mutation diminishing Orb2A's prion-like oligomerization forms long-term memory but fails to maintain it over time. Since, this prion-like oligomerization of Orb2A plays a crucial role in the maintenance of memory, here we aim to find what regulates this oligomerization. In an immunoprecipitation-based screen, we identify interactors of Orb2A in the Hsp40 and Hsp70 families of proteins. Amongst these, we find an Hsp40 family protein Mrj as a regulator of the conversion of Orb2A to its prion-like form. Mrj interacts with Hsp70 proteins and acts as a chaperone by interfering with the aggregation of pathogenic Huntingtin. Unlike its mammalian homolog, we find Drosophila Mrj is neither an essential gene nor causes any gross neurodevelopmental defect. We observe a loss of Mrj results in a reduction in Orb2 oligomers. Further, the knockdown of Mrj in the mushroom body neurons results in a deficit in long-term memory. Our work implicates a chaperone Mrj in mechanisms of memory regulation through controlling the oligomerization of Orb2A and its association with the translating polysomes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.10.511666v1?rss=1 Authors: Karasmanis, E. P., Reimer, J. M., Kendrick, A. A., Rodriguez, J. A., Truong, J. B., Lahiri, I., Reck-Peterson, S. L., Leschziner, A. E. Abstract: Cytoplasmic dynein-1 transports many intracellular cargos towards microtubule minus ends. Dynein is autoinhibited and undergoes conformational changes to form an active complex, consisting of one or two dynein dimers, the dynactin complex and activating adaptor(s)1,2. The Lissencephaly 1 gene, LIS1, is genetically linked to the dynein pathway from fungi to mammals and is mutated in patients with the neurodevelopmental disease lissencephaly3-5. Lis1 is required for active dynein complexes to form6-10, but how it does so is unclear. Here, we present a structure of two dynein motor domains with two Lis1 dimers wedged in-between. The contact sites between dynein and Lis1 in this structure, termed "Chi", are required for Lis1s regulation of dynein in Saccharomyces cerevisiae in vivo and the formation of active human dynein-dynactin- activating adaptor complexes in vitro. We propose that this structure represents an intermediate in dyneins activation pathway, revealing how Lis1 relieves dyneins autoinhibited state. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

This week we discuss how cells can divide without nuclei and why this happens with Anand Bakshi, Fabio Echegaray Iturra (@fabioechegaray) & Mustafa G. Aydogan @AydoganLab. We also highlight Drosophila as an amazing model system that just keeps on giving and speak about the importance of preprints in collaboration and grant applications. We also discuss the limitations of preprints and the fluidity of science. Read the full preprint: https://www.biorxiv.org/content/10.1101/2022.06.15.496343v1.full Other links: https://www.theguardian.com/science/2017/oct/07/fruit-fly-fascination-nobel-prizes-genetics https://aydoganlab.com/ This episode was produced by Emma Wilson and edited by Jonny Coates. If you enjoyed this show then hit that subscribe button and leave a review (on Apple Podcasts or Spotify). If you love what we are trying to do then buy us a coffee https://www.buymeacoffee.com/preprints! Any contribution is greatly appreciated. For the latest podcast news and updates follow us on Twitter @MotionPod or visit our website; www.preprintsinmotion.com. Produced by JEmJ Productions (find us on Twitter: Jonny @JACoates, Emma @ELWilson92, John @JohnDHoward8) and generously supported by ASAPbio (https://asapbio.org | @asapbio_).

Research on the Wolbachia bacteria is applied to the malaria-transmitting Anopheles mosquito, with exciting implications for vector control. Transcript New tools are needed to control the Anopheles mosquito, the vector of malaria. Bacteria called Wolbachia might be the answer. Although Aedes mosquitoes do not naturally carry Wolbachia, when the bacteria were introduced from other sources, they could no longer transmit pathogenic viruses, like dengue. Harnessing this technology to control vector populations is now being applied to the malaria-transmitting Anopheles mosquito. New research has shown that ‘cytoplasmic incompatibility' [CI] can be induced in Anopheles by expressing the Wolbachia cifB gene in male mosquitoes, and that this lethality can be reversed by expression of the cifA gene. In other words, when male mosquitoes mate with females that do not carry the bacteria, they cannot produce offspring. Knowing this will help pave the way for the use of Wolbachia to control malaria mosquitoes in the field. Source Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector About The Podcast The Johns Hopkins Malaria Minute podcast is produced by the Johns Hopkins Malaria Research Institute to highlight impactful malaria research and share with the global community.

In this episode of Curbside Consults, we examine the latest guidelines on the management of ANCA-associated vasculitis by the American College of Rheumatology (ACR) and Vasculitis Foundation. In doing so, we review the major trials in vasculitis research published during the last 10 years with one of the guideline authors, Dr. Peter...

What is nuclear cytoplasmic asynchrony and why does it happen. In this episode, we will talk about the pathomechanism of nuclear cytoplasmic asynchrony. --- This episode is sponsored by · Anchor: The easiest way to make a podcast. https://anchor.fm/app

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.374231v1?rss=1 Authors: Blumenstock, S., Schulz-Trieglaff, E. K., Bolender, A.-L., Voelkl, K., Lapios, P., Lindner, J., Hipp, M. S., Hartl, U., Klein, R., Dudanova, I. Abstract: The cellular protein quality control machinery is important for preventing protein misfolding and aggregation, and decline in protein homeostasis (proteostasis) is believed to play a crucial role in age-related neurodegenerative disorders. However, how proteostasis capacity of neurons changes in different diseases is not yet sufficiently understood, and progress in this area has been hampered by the lack of tools to monitor proteostasis in mammalian models. Here, we have developed reporter mice for in vivo analysis of neuronal proteostasis. The mice express EGFP-fused firefly luciferase (Fluc), a conformationally unstable protein that requires chaperones for proper folding and sensitively reacts to proteotoxic stress by formation of intracellular Fluc-EGFP foci and by reduced luciferase activity. Using these mice, we provide evidence for proteostasis decline in the aging brain. Moreover, we find a marked impairment in proteostasis in tauopathy mice, but not in Huntington's disease mice. Mechanistic investigations in primary neuronal cultures demonstrate that cytoplasmic, but not nuclear, aggregates cause defects of cellular protein quality control. Thus, the Fluc-EGFP reporter mice enable new insights into proteostasis alterations in different diseases. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.25.354399v1?rss=1 Authors: Quek, H., Cuni-Lopez, C., Stewart, R., Colletti, T., Notaro, A., Sun, Y., Guo, C. C., Lupton, M. K., Nguyen, T. H., Oikari, L. E., Roberts, T. L., Lim, Y. C., La Bella, V., White, A. R. Abstract: Aims: Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterised by the loss of upper and lower motor neurons. Neuroinflammation mediated by microglial activation is evident in post-mortem brain tissues, and in brain imaging of patients with ALS. However, the exact role of microglia in ALS remains to be elucidated partly due to the lack of an accurate microglial model system that is able to recapitulate the clinical pathology of ALS. Moreover, direct sampling of microglia from patients with ALS is not feasible, further limiting the study of microglial function in ALS. To address this shortcoming, we describe an approach that generates monocyte-derived microglia (MDMi) that are capable of expressing molecular markers, and functional characteristics similar to resident human brain microglia. Importantly, MDMi can be routinely and reproducibly generated from ALS patient blood, and reveal patient heterogeneity associated with age, sex and disease subgroup. Methods: MDMi were successfully established from all 30 ALS patients, including 15 patients with slow disease progression, 6 with intermediate progression, and 9 with rapid progression, together with 20 non-affected heathy controls (HC). Results: Our ALS MDMi model recapitulated canonical pathological features of ALS including non-phosphorylated and phosphorylated-TDP-43-positive pathological inclusions. We further observed significantly impaired phagocytosis, altered cytokine expression and microglial morphology, as well as elevated DNA damage in ALS compared to HC MDMi. Abnormal phagocytosis was observed in all ALS cases, and was correlated to the progression of disease. Moreover, in-depth analysis of individual microglia revealed cell-specific variation in phagocytic function that was significantly altered, and exacerbated in rapid disease progression. Conclusions: Our approach enabled us to generate ALS patient microglia from peripheral blood samples using a rapid, robust, cost-effective, and reproducible protocol. We have shown that ALS monocyte-derived microglia have significantly altered functional behaviour compared to age-matched HCs, with a major deficit in phagocytic activity. This is also the first demonstration of abnormal TDP-43 localisation in microglia grown from ALS patients. Overall, this approach is highly applicable to monitor disease progression and can be applied as a functional readout in clinical trials for anti-neuroinflammatory agents. Additionally, this model system can be used as a basis for personalised therapeutic treatment for ALS, as well as other neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.06.327742v1?rss=1 Authors: Kliche, J., Ali, M., Ivarsson, Y. Abstract: The spike protein of the SARS-CoV-2 interacts with angiotensin converting enzyme 2 (ACE2) and enters the host cell by receptor-mediated endocytosis. Concomitantly, evidence is pointing to the involvement of additional host cell receptors, such as integrins. The cytoplasmic tails of ACE2 and integrin beta3 contain a plethora of predicted binding motifs. Here, we confirm the functionality of some of these motifs through affinity measurements. The class I PDZ binding motif in the ACE2 cytoplasmic tail binds the first PDZ domain of the scaffold protein NHERF3. The clathrin-adaptor subunit AP2 Mu2 interacts with an endocytic motif in the ACE2 with low affinity and the interaction is abolished by phosphorylation of Tyr781. Furthermore, the C-terminal region of integrin beta3 contains a LC3-interacting region, and its interaction with ATG8 domains is enhanced by phosphorylation. Together, our data provides possible molecular links between host cell receptors and endocytosis and autophagy Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.20.305243v1?rss=1 Authors: Gennerich, A., Liu, X., Rao, L. Abstract: Cytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein's mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein's stepping cycle and underscores the complexity and crosstalk among the motor's multiple AAA+ domains. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.297036v1?rss=1 Authors: Frottin, F., Perez-Berlanga, M., Hartl, F.-U., Hipp, M. S. Abstract: The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate, DPRs exert additive effects that may contribute to pathology. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.27.270991v1?rss=1 Authors: Jin, F., Sun, M., Fujii, T., Yamada, Y., Wang, J., Maturana, A., Wada, M., Su, S., Ma, J., Takeda, H., Kusakizako, T., Tomita, A., Nakura, Y. N., Liu, K., Uemura, T., Nomura, Y., Nomura, N., Ito, K., Nureki, O., Namba, K., Iwata, S., Yu, Y., Hattori, M. Abstract: MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.30.228270v1?rss=1 Authors: Imakubo, M., Takayama, J., Okada, H., Onami, S. Abstract: Background: Oocyte quality decreases with aging, thereby increasing errors in fertilization, chromosome segregation, and embryonic cleavage. Oocyte appearance also changes with aging, suggesting a functional relationship between oocyte quality and appearance. However, no methods are available to objectively quantify age-associated changes in oocyte appearance. Results: We show that statistical image processing of Nomarski differential interference contrast microscopy images can be used to quantify age-associated changes in Caenorhabditis elegans oocyte appearance. Max-min Value (mean difference between the maximum and minimum intensities within each moving window) quantitatively characterized the difference in oocyte cytoplasmic texture between 1- and 3-day-old adults (Day 1 and Day 3 oocytes, respectively). With an appropriate parameter set, the gray level co-occurrence matrix (GLCM)-based texture feature Correlation (COR) more sensitively characterized this difference than the Max-min Value. Manipulating the smoothness of and/or adding irregular structures to the cytoplasmic texture of Day 1 oocyte images reproduced the difference in Max-min Value but not in COR between Day 1 and Day 3 oocytes. Increasing the size of granules in synthetic images recapitulated the age-associated changes in COR. Manual measurements validated that the cytoplasmic granules in oocytes become larger with aging. Conclusions: The Max-min Value and COR objectively quantify age-related changes in C. elegans oocyte in Nomarski DIC microscopy images. Our methods provide new opportunities for understanding the mechanism underlying oocyte aging. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.29.226977v1?rss=1 Authors: Riguet, N., Mahul-Mellier, A.-L., Maharjan, N., Burtscher, J., Patin, A., Croisier, M., Knott, G. W., Reiterer, V., Farhan, H., Lashuel, H. Abstract: Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington's disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt inclusions in cells overexpressing mutant exon1 of the Htt protein. Our findings provide novel insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve 1) polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; 2) recruitment and accumulation of remodeled or dysfunctional membranous organelles; and 3) impairement of the protein quality control and degradation machineries. Interestingly, nuclear and cytoplasmic Htt inclusions exhibited distinct biochemical composition and ultrastructural properties, suggesting different formation mechanisms and toxicity. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.20.211557v1?rss=1 Authors: Tyzack, G. E., Neeves, J., Klein, P., Crerar, H., Ziff, O., Taha, D. M., Luisier, R., Luscombe, N. M., Patani, R. Abstract: Intron retention is known to regulate gene expression. We recently described intron retention as the predominant splicing programme characterizing early stages of motor neurogenesis from human induced pluripotent stem cells (hiPSCs) and it's perturbation in familial amyotrophic lateral sclerosis (ALS). Here, we sought to gain more insight into the nucleocytoplasmic distribution of aberrant intron-retaining transcripts (IRTs) and to identify their discriminating molecular features. We combined cellular fractionation with hiPSCs undergoing motor neurogenesis and deep-sequenced 95 samples: ALS vs control hiPSCs and nuclear vs cytoplasmic compartments across six timepoints, which represents a rich transcriptomic resource for basic and applied neuroscientists. Using this resource, we identified >100 aberrant cytoplasmic IRTs in cultures carrying ALS-causing VCP gene mutations. We taxonomized aberrant IRTs by their nucleocytoplasmic distribution and demonstrate that these classes exhibit sequence-specific attributes and differential predicted binding affinity to ALS-related RNA binding proteins. In summary we uncover a distinct class of cytoplasmic IRTs that serve as blueprints for established molecular hallmarks of ALS (nuclear-to-cytoplasmic mislocalisation of TDP-43, SFPQ and FUS) and therefore may also represent therapeutic targets. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.29.178160v1?rss=1 Authors: Rahmati, N., Normoyle, K. P., Glykys, J., Dzhala, V. I., Lillis, K. P., Kahle, K. T., Raiyyani, R., Jacob, T., Staley, K. J. Abstract: Developmental, cellular, and subcellular variations in the direction of neuronal Cl- currents elicited by GABAA receptor activation have been frequently reported, and we found a corresponding variance in the reversal potential (EGABA) for individual interneurons synapsing on a single pyramidal cell. These findings suggest a corresponding variance in the cytoplasmic concentration of Cl- ([Cl-i]). We determined [Cl-]i by: 1) two-photon imaging of the Cl- sensitive, ratiometric fluorescent protein SuperClomeleon (sCLM); 2) Fluorescence Lifetime IMaging (FLIM) of the Cl- sensitive fluorophore MEQ; and 3) electrophysiological measurements of EGABA. These methods collectively demonstrated stable [Cl-]i microdomains in individual neurons in vivo. Fluorometric and electrophysiological estimates of local [Cl-]i were highly correlated. [Cl-]i microdomains persisted after pharmacological inhibition of cation-chloride cotransporters (CCCs) but steadily decreased after inhibiting the polymerization of the anionic macromolecule actin. These studies highlight the existence of functionally significant neuronal Cl- microdomains that modify the impact of GABAergic inputs. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.09.141556v1?rss=1 Authors: Scekic-Zahirovic, J., Sanjuan-Ruiz, I., Kan, V., Megat, S., de Rossi, P., Dieterle, S., Cassel, R., Kessler, P., Wiesner, D., Tzeplaeff, L., Demais, V., Mueller, H.-P., Picchiarelli, G., Mishra, N., Dirrig-Grosch, S., Kassubek, J., Rasche, V., Ludolph, A. C., Boutillier, A.-L., Polymenidou, M., Lagier-Tourenne, C., Liebscher, S., Dupuis, L. Abstract: Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a profound increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo. Importantly, RNAseq analysis suggested involvement of defects in inhibitory neurons, that was confirmed by ultrastructural and morphological defects of inhibitory synapses and increased synaptosomal levels of mRNAs involved in inhibitory neurotransmission. Thus, cytoplasmic FUS triggers inhibitory synaptic deficits, leading to increased neuronal activity and behavioral phenotypes. FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, but also in other neurodegenerative diseases with FUS mislocalization. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.16.100032v1?rss=1 Authors: Ford, L., Asok, A., Tripp, A. D., Parro, C., Fitzpatrick, M., de Solis, C. A., Shafiian, N., Fioriti, L., Soni, R. K., Kandel, E. Abstract: Biomolecular condensates, membraneless organelles found throughout the cell, play critical roles in many aspects of cellular function. Ribonucleoprotein granules (RNPs), a type of biomolecular condensate found in neurons that are necessary for local protein synthesis and are involved in long-term potentiation (LTP). Several RNA-binding proteins present in RNPs are necessary for the synaptic plasticity involved in LTP and long-term memory. Most of these proteins possess low complexity motifs, allowing for increased promiscuity. We explore the role the low complexity motif plays for RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CPEB3), a protein necessary for long-term memory persistence. We found that RNA binding and SUMOylation are necessary for CPEB3 localization to the P body, thereby having functional implications on translation. Here, we investigate the role of the low complexity motif of CPEB3 and find that it is necessary for P body localization and downstream targeting for local protein synthesis. Copy rights belong to original authors. Visit the link for more info

In this Episode we sat down with Shelley Berger, Keynote Speaker at the "EMBO | EMBL Symposium: Metabolism Meets Epigenetics" to talk about her work on Epigenetic Mechanisms of Aging and Longevity. On how cytoplasmic chromatin fragments are involved in these processes, how alcohol has an effect on Histone PTMs in the brain and last but not least how Ants became her favorite Model Organism. References Hazel A. Cruickshanks, Tony McBryan, … Peter D. Adams (2013) Senescent cells harbour features of the cancer epigenome (Nature Cell Biology) DOI: 10.1038/ncb2879 Zhixun Dou, Kanad Ghosh, … Shelley L. Berger (2017) Cytoplasmic chromatin triggers inflammation in senescence and cancer (Nature) DOI: 10.1038/nature24050  Hua Yan, Comzit Opachaloemphan, … Claude Desplan (2017) An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants (Cell) DOI: 10.1016/j.cell.2017.06.051  P. Mews, G. Egervari, … S. L. Berger (2019) Alcohol metabolism contributes to brain histone acetylation (Nature) DOI: 10.1038/s41586-019-1700-7  Karl M. Glastad, Riley J. Graham, … Shelley L. Berger (2019) Epigenetic Regulator CoREST Controls Social Behavior in Ants (Molecular Cell) DOI: 10.1016/j.molcel.2019.10.012 Contact https://twitter.com/activemotif https://twitter.com/epigenetics_pod https://www.linkedin.com/company-beta/35651/ https://www.facebook.com/ActiveMotifInc/ podcast@activemotif.com

The complete TWiV team give a report on the Ebola virus outbreak in DRC, and reveal that cell surface nectin proteins cause the transfer of cytoplasmic cargo, including measles virus, between cells. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Fire Alarm at ASV Ebola outbreak sitrep(WHO) Ebola outbreak dashboard(WHO) Ebola outbreakDRC (MSF) Ebola articles at CIDRAP Ebola virus vaccine could run out(STAT news) Nectin mediated cytoplasmic transfer(J Cell Sci) Letters readon TWiV 559 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - DukeMed Distinguished Award: Bill Joklik Alan - Audubon photography awards for 2019 Rich - Joklik paper on vaccinia virus mRNA; Joklik MEM Dickson- Ghost orchid pollination revealed Kathy- Crocheted petri plates Vincent- Documents relating to history of ASV Listener Picks Neva - What happens if we engineer a superbug? Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The complete TWiV team give a report on the Ebola virus outbreak in DRC, and reveal that cell surface nectin proteins cause the transfer of cytoplasmic cargo, including measles virus, between cells. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, Kathy Spindler, and Brianne Barker Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode Fire Alarm at ASV Ebola outbreak sitrep(WHO) Ebola outbreak dashboard(WHO) Ebola outbreakDRC (MSF) Ebola articles at CIDRAP Ebola virus vaccine could run out(STAT news) Nectin mediated cytoplasmic transfer(J Cell Sci) Letters readon TWiV 559 Timestamps by Jolene. Thanks! Weekly Science Picks Brianne - DukeMed Distinguished Award: Bill Joklik Alan - Audubon photography awards for 2019 Rich - Joklik paper on vaccinia virus mRNA; Joklik MEM Dickson- Ghost orchid pollination revealed Kathy- Crocheted petri plates Vincent- Documents relating to history of ASV Listener Picks Neva - What happens if we engineer a superbug? Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

We often imagine a cell as a large balloon filled with jelly, but really it is more like a large city. Packages need to go from one place to the other in an organized fashion as to not disrupt other processes. For example, when we need an item, we go to the store or click away on retail websites, but how do these items find their way to the retail place or our house? There are vehicles on roads and highways that are utilized for distribution. Much like the infrastructure that we use everyday to move cargo around our cities, the cell has its own system to deliver goods from one place to another. What are the 18 wheelers of the cell, how do they move such important packages, and how do they know where to go? Cytoplasmic dynein is a protein complex that transports molecular cargo along and plays a key role in the intracellular trafficking network. Dr. Danielle Grotjahn utilizes specialized imaging techniques to study these structures and the function of motor proteins.

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler The TWiVirions reveal bacteriophage genes that control eukaryotic reproduction, and the biochemical basis for increased Ebolavirus glycoprotein activity during the recent outbreak.   Become a patron of TWiV! Links for this episode Nido2017 Meeting ASM Microbe 2017 Mark Buller obituary RS virus pipeline (pdf) Prophage WO genes and cytoplasmic incompatibility (Nature) Vanderbilt Virology (TWiV 332) Basis for increased Ebolavirus glycoprotein activity (Cell Host Micr) Letters read on TWiV 431 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twiv Weekly Science Picks Rich - Germ Theory by Robert P. GaynesAlan - Student Scientist Partnerships Kathy - Symbiartic Dickson - PaleoBioDB Navigator Vincent - Kusama Infinity Room Listener Pick Chaim - How to fight back against the backfire effect Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Frontotemporal dementia is the second most common neurodegenerative disease in people younger than 65 years. Patients suffer from behavioral changes, language deficits and speech impairment. Unfortunately, there is no effective treatment available at the moment. Cytoplasmic inclusions of the DNA/RNA-binding protein TDP-43 are the pathological hallmark in the majority of FTLD cases, which are accordingly classified as FTLD-TDP. Mutations in GRN, the gene coding for the trophic factor progranulin, are responsible for the majority of familiar FTLD-TDP cases. The first genome-wide association study performed for FTLD-TDP led to the identification of risk variants in the so far uncharacterized gene TMEM106B. Initial cell culture studies revealed intracellular localization of TMEM106B protein in lysosomes but its neuronal function remained elusive. Based on these initial findings, I investigated the physiological function of TMEM106B in primary rat neurons during this thesis. I demonstrated that endogenous TMEM106B is localized to late endosomes and lysosomes in primary neurons, too. Notably, knockdown of the protein does neither impair general neuronal viability nor the protein level of FTLD associated proteins, such as GRN or TDP-43. However, shRNA-mediated knockdown of TMEM106B led to a pronounced withering of the dendritic arbor in developing and mature neurons. Moreover, the strong impairment of dendrite outgrowth and maintenance was accompanied by morphological changes and loss of dendritic spines. To gain mechanistic insight into the loss-of-function phenotypes, I searched for coimmunoprecipitating proteins by LC-MS/MS. I specifically identified the microtubule-binding protein MAP6 as interaction partner and was able to validate binding. Strikingly, overexpression of MAP6 in primary neurons phenocopied the TMEM106B knockdown effect on dendrites and loss of MAP6 restored dendritic branching in TMEM106B knockdown neurons, indicating functional interaction of the two proteins. The link between a lysosomal and a microtubule-binding protein made me study the microtubule dependent transport of dendritic lysosomes. Remarkably, live cell imaging studies revealed enhanced movement of dendritic lysosomes towards the soma in neurons devoid of TMEM106B. Again, MAP6 overexpression phenocopied and MAP6 knockdown rescued this effect, strengthening the functional link. The MAP6-independent rescue of dendrite outgrowth by enhancing anterograde lysosomal movement provided additional evidence that dendritic arborization is directly controlled by lysosomal trafficking. From these findings I suggest the following model: TMEM106B and MAP6 together act as a molecular brake for the retrograde transport of dendritic lysosomes. Knockdown of TMEM106B and (the presumably dominant negative) overexpression of MAP6 release this brake and enhance the retrograde movement of lysosomes. Subsequently, the higher protein turnover and the net loss of membranes in distal dendrites may cause the defect in dendrite outgrowth. The findings of this study suggest that lysosomal misrouting in TMEM106B risk allele carrier might further aggravate lysosomal dysfunction seen in patients harboring GRN mutations and thereby contribute to disease progression. Taken together, I discovered the first neuronal function for the FTLD-TDP risk factor TMEM106B: This lysosomal protein acts together with its novel, microtubule-associated binding partner MAP6 as molecular brake for the dendritic transport of lysosomes and thereby controls dendrite growth and maintenance.

Integrins are ubiquitously expressed adhesion receptors with important functions in cellular adhesion, proliferation, migration and signaling. These functions are determined by integrin trafficking through endosomal compartments and receptor affinity regulation. In this thesis, we identified the distal NxxY motif of the β1 integrin cytoplasmic tail as a molecular switch modulating a spatiotemporally controlled binding of two FERM-domain proteins in different cellular compartments. Kindlins mediate integrin activation at the plasma membrane and they dislodge upon internalization. In the endosomal compartment, the free cytoplasmic domain is subsequently bound by sorting nexin 17 (SNX17) to inhibit integrin degradation. We identified SNX17 as a new β1 integrin adaptor protein, which uses the kindlin-binding site in endosomal compartments to stabilize integrins and to promote their recycling back to the plasma membrane.

Vincent, Elio and Michael recorded this episode of This Week in Microbiology before an audience at the 2013 General Meeting of the American Society for Microbiology in Denver, Colorado, where they spoke with Andrew Camilli, Ferric Fang, Suzanne Fleiszig, and Michelle Swanson about their research on a phage system for evading innate immunity, retractions of research papers, bacterial infections of the eye, and cytoplasmic defenses against intracellular bacteria.

Vincent, Elio and Michael recorded this episode of This Week in Microbiology before an audience at the 2013 General Meeting of the American Society for Microbiology in Denver, Colorado, where they spoke with Andrew Camilli, Ferric Fang, Suzanne Fleiszig, and Michelle Swanson about their research on a phage system for evading innate immunity, retractions of research papers, bacterial infections of the eye, and cytoplasmic defenses against intracellular bacteria.

Vincent, Elio and Michael recorded this episode before an audience at the 2013 General Meeting of the American Society for Microbiology in Denver, Colorado, where they spoke with Andrew, Ferric, Suzanne, and Michelle about their research on a phage system for evading innate immunity, retractions of research papers, bacterial infections of the eye, and cytoplasmic defenses against intracellular bacteria. This episode was filmed live at ASM GM 2013 in Denver, CO. Visit www.microbeworld.org/asmlive to watch the full video archive of this episode as well as all the videos recorded during GM. 

Vincent, Elio and Michael recorded this episode of This Week in Microbiology before an audience at the 2013 General Meeting of the American Society for Microbiology in Denver, Colorado, where they spoke with Andrew Camilli, Ferric Fang, Suzanne Fleiszig, and Michelle Swanson about their research on a phage system for evading innate immunity, retractions of research papers, bacterial infections of the eye, and cytoplasmic defenses against intracellular bacteria.

Andrea D'ambrogio MRC Laboratory of Molecular Biology, Cambridge, UK speaks on "The Role of Cytoplasmic Polyadenylation Proteins in Translation, miRNA stability and Cancer". This seminar has been recorded by ICGEB Trieste

Thu, 1 Mar 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16103/ https://edoc.ub.uni-muenchen.de/16103/1/Heym_Roland_G.pdf Heym, Roland Gerhard ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Learn about an unexpected pro-apoptotic role for the retinoblastoma tumor suppressor (start 1:22), a cytoplasmic filter in neurons (start 9:35) and an intricate molecular interaction that underpins the oxidative stress response in yeast (start 17:13).

The extracellular matrix (ECM) provides the structural frame for the development of tissues and organs. The ECM is bound by numerous membranous matrix-adhesion molecules and thereby triggers intracellular signals that control various cellular functions such as survival, polarity, proliferation and differentiation. Integrins represent an important family of ECM adhesion molecules which link the ECM with the intracellular actin-cytoskeleton. Integrin mediated adhesion structures also serve as important signaling platforms, although the integrin itself does not harbors any catalytic domains. Therefore integrin signaling depends on the recruitment of a number of cytoplasmic proteins that directly or indirectly bind to the short cytoplasmic integrin tails. During my PhD thesis I worked on three of these molecules, ILK, Kindlins and Palladin, and used the mouse as a model system to address their in vivo function. First, I investigated the role of integrin-linked kinase (ILK) in skeletal muscle. Loss of ILK expression in mice leads to peri-implantation lethality due to a cell polarization defect of the early embryo and abnormal actin accumulations. Studies in Caenorhabditis elegans and Drosophila melanogaster revealed an essential function for ILK in the attachment of actin filaments to the membrane of muscle cells and lack of ILK expression results in early lethality during embryogenesis. We generated mice with a skeletal muscle-restricted deletion of ILK that developed a mild, but progressive muscular dystrophy. This phenotype is predominantly restricted to myotendinous junctions (MTJs). Ultrastructural analyses showed muscle cell detachment from the basement membranes, and an accumulation of extracellular matrix. Endurance exercise training enhances the defect leading to disturbed subsarcolemmal myofiber architecture and an abrogation of the phosphorylation of Ser473 as well as Thr308 of protein kinase B (PKB)/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Second, I studied the expression and in vivo function of a further integrin- and actin- associated protein, palladin. Palladin belongs to the palladin/myotilin/myopalladin protein family. Palladin represents a phosphoprotein which plays an important role in cell adhesion and motility. Initially, I characterized the gene structure and the expression pattern of palladin. The palladin gene spans about 400 kb, with 25 exons and 3 alternative promoters resulting in at least three different isoforms (200 kDa, 140 kDa and 90-92 kDa) in mice. Using RT-PCR and in situ hybridizations of embryonic and adult tissues, I could show that the 200kDa isoform is predominantly expressed in heart and skeletal muscle. In contrast, the 140kDa isoform is expressed in various tissues and represents the major palladin isoform of the brain. The 90-92 kDa isoform is almost ubiquitously expressed with highest levels in tissues rich in smooth muscle, like bladder, uterus, small intestine and colon. The expression of the 200kDa isoform was characterized in more detail with a polyclonal antibody showing that this isoform localizes to the Z-discs of heart and skeletal muscle cells. In vitro differentiation experiments with a mouse myoblast cell line revealed an induction of the 200kDa isoform during myoblast fusion and differentiation suggesting that the biggest palladin isoform may serve as a molecular scaffold during myogenesis. Third, I specifically inactivated the largest palladin isoform in mice. Lack of the 200 kDa palladin isoform has no impact on the development, viability and fertility of mice. However ultrastructural analyses by transmission electron microscopy (TEM) showed a mild cardiac myopathy due to disintegration of myofibrils. In collaboration with the group of Olli Carpén, we generated palladin 200 kDa isoform/ myotilin double knockout mice. Myotilin is also expressed in heart and skeletal muscle. Ablation of both myotilin and palladin 200 kDa isoform in mouse revealed in addition to the mild cardiac myopathy a structural and functional impairment of skeletal muscle. Finally, I was also involved in the characterization of the expression and subcellular localization of a novel family of integrin associated proteins: the Kindlins. The Kindlin family consists of three members, Kindlin-1, -2 and -3. Mutations in Kindlin-1 cause a human disease, called Kindler Syndrome, which represents a skin blistering disease affecting the actin cytoskeleton of basal keratinocytes. Kindlin gene expression was first analyzed at the mRNA level by RT-PCR and Northern Blot studies. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia. Kindlin-2 is expressed in all tissues with highest levels in striated and smooth muscle cells. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. In contrast, Kindlin-3 expression is restricted to hematopoietic cells. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in podosomes, which are specialized adhesion structures of hematopoietic cells.

Messenger RNA localization occurs in the cytoplasm and allows temporal and spatial regulation of gene expression. In yeast, the localization of ASH1 mRNA to the tip of budding cells allows the asymmetric sorting of Ash1 protein, which has a key function in the regulation of mating-type switching. After cell division, asymmetric distribution of Ash1p restricts mating type switching to only the mother cell. The cytoplasmic transport of ASH1 mRNA to the bud tip depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. Three additional trans-acting factors Khd1p, Puf6p and Loc1p are involved in this process. All known RNA-binding proteins of ASH1 mRNA have revealed a nuclear connection, when following their cellular distribution by indirect immunofluorescence. Thus, an early step in the localization pathway might be the early recruitment of specific trans-acting factors to the mRNA already in the nucleus. The aim of this thesis was to investigate how nuclear key events such as early binding to localized transcripts and the subsequent assembly into a nuclear RNP can account for effective RNA localization. Following the route of She2p, it was possible to show nucleo-cytoplasmic shuttling of this RNA binding protein. Moreover, ASH1 mRNA and She2p were found accumulated within the nucleolus upon arrest of mRNA export. Interestingly, two additional trans-acting factors, Loc1 and Puf6p, both involved in ASH1 mRNA localization are also nucleolar proteins. Moreover, She2’s nuclear history seems to be important for an effective sorting of Ash1p. When restricting ASH1-She2p association to the cytoplasmic compartment artificially, the ASH1 mRNA was still localized but was prematurely translated during its transport. This suggests that nuclear RNP assembly has an influence on the later stages of cytoplasmic translational control. The nucleolus might represent the appropriate cellular compartment to provide the spatial framework for the assembly of localization competent RNPs since many RNPs are assembled in this region.

HYPERGLYCAEMIA impairs recovery from transient cerebral ischaemia: the importance of tissue acidification for this phenomenon has not been clarified in detail. We investigated this issue in a less complex in vitro preparation of isolated rat dorsal spinal roots exposed for 30 min to hyperglycaemic hypoxia. Peak height of compound action potentials recovered minimally in 5 mM bicarbonate. However, recovery was greatly improved by addition of the weak base trimethylamine during re-oxygenation. Addition of the weak acid propionate had no such effect. Cytoplasmic alkalinization improved recovery in a brief time window only: application of trimethylamine after 15 min of re-oxygenation was without beneficial effect. These data emphasize the importance of cytoplasmic acidification for neurophysiological recovery from hyper-glycaemic hypoxia during the initial period of re-oxygenation.

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals.

Tue, 1 Jan 1991 12:00:00 +0100 http://epub.ub.uni-muenchen.de/8687/ http://epub.ub.uni-muenchen.de/8687/1/Mueller_Guenter_8687.pdf Müller, Günter; Bandlow, Wolfhard Müller, Günter und Bandlow, Wolfhard (1991): Two lipid-anchored cAMP-binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A. In: European Journal of Biochemistry, Vol. 202, Nr. 2: pp.

Thu, 1 Jan 1976 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7307/1/7307.pdf Rücker, Axel von; Neupert, Walter

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl2. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of sim270 x 210 x 215 Å; cytoplasmic ribosomes measure sim295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is sim130 Å, while the diameter of cytoplasmic ribosomes is sim 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.

Wed, 1 May 1974 12:00:00 +0100 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-44W4WF3-3F&_user=616146&_coverDate=05%2F01%2F1974&_rdoc=21&_fmt=high&_orig=browse&_srch=doc-info(%23toc%234938%231974%23999589997%23280309%23FLP%23display%23Volume)&_cdi=4938&_sort=d&_docancho https://epub.ub.uni-muenchen.de/7268/1/Neupert_Walter_7268.pdf Neupert, Walter; Hallermayer, Gerhard

Tue, 1 Jan 1974 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7274/1/7274.pdf Neupert, Walter

Tue, 1 Jan 1974 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7281/1/7281.pdf Neupert, Walter; Kleinow, W.; Miller, Fritz